A20 or tumor necrosis factor (TNF)-induced protein 3 (TNFAIP3) is a negative regulator of nuclear factor-kappaB (NF-kappaB). We have investigated whether polymorphisms in this gene are associated with increased atherosclerosis in diabetic patients. Five tag single nucleotide polymorphisms (SNPs) were typed in 479 type 2 diabetic patients from Boston, including 239 coronary artery disease (CAD)-positive case subjects and 240 CAD-negative control subjects. Two tag SNPs (rs5029930 and rs610604) were independently associated with CAD; adjusted odds ratios (ORs) for minor allele carriers were 2.3 (95% CI 1.4-3.8, P = 0.001) and 2.0 (1.3-2.9, P = 0.0008), respectively. The association with rs610604 was dependent on glycemic control, with ORs of 3.9 among subjects with A1C < or =7.0% and 1.2 for those with A1C >7.0% (P for interaction = 0.015). A similar interaction pattern was found among 231 CAD-positive and 332 CAD-negative type 2 diabetic patients from Italy (OR 2.2, P = 0.05 vs. OR 0.9, P = 0.63 in the low vs. high A1C strata, P for interaction = 0.05). Quantitative RT-PCR in blood mononuclear cells from 83 nondiabetic subjects showed that rs610604 and rs5029930 minor allele homozygotes have 30-45% lower levels of A20 mRNA than major allele homozygotes, and heterozygotes have intermediate levels (P = 0.04 and 0.028, respectively). These findings point to variability in the A20/TNFAIP3 gene as a modulator of CAD risk in type 2 diabetes. This effect is mediated by allelic differences in A20 expression.